Encoding multiple virtual signals in DNA barcodes with single-molecule FRET

DNA barcoding provides a way to label a huge number of different biological molecules using the extreme programmability in DNA sequence synthesis. Fluorescence imaging is an easy−to−access method to detect individual DNA barcodes, which can be scaled up to a massively high−throughput format. Large overlaps between emission spectra of fluorescence dyes, however, severely limit the numbers of DNA barcodes−and thus its signal space−that can be detected in a simultaneous manner. We here demonstrate the use of single−molecule fluorescence resonance energy transfer (FRET) to encode virtual signals in DNA barcodes using conventional two−color fluorescence microscopy. By optimizing imaging and biochemistry conditions for weak hybridization events for DNA barcodes, we markedly enhanced accuracy in our determination of the efficiency by which single−molecule FRET occurred, reaching an error less than 0.01 in the FRET efficiency domain. This allowed us to unambiguously differentiate six DNA barcodes exhibiting different FRET values without involving probe sequence exchanges. Our method can be directly incorporated with previous DNA barcode techniques, and may thus be widely adopted to expand the signal space of the DNA barcode techniques.